构建永生化的人骨髓间充质干细胞
发布时间:2018-09-02 08:56
【摘要】: 目的:构建永生化的人骨髓间充质干细胞。 方法:采用阳离子脂质体介导法将病毒质粒pBabepuro-hTERT导入包装细胞内,嘌呤霉素(Puro)筛选出病毒表达阳性的细胞克隆,测定病毒滴度。取培养病毒的上清液感hBMSCs细胞、筛选出转基因细胞,我们将其命名为hTERT-hBMSCs细胞。采用端粒酶重复序列扩增(telomere repe at amplification protocol assay ,TRAP)酶链免疫吸附试验(ELISA)法检测hBMSCs细胞转染前后端粒酶活性的变化;采用MTT法(四甲基偶氮唑盐微量酶反应比色法)测绘转染前后细胞生长曲线,观察hTERT转入后对细胞增殖的影响;克隆形成实验检测hTERT-hBMSCs细胞的增殖能力;染色体核型分析观察hTERT-hBMSCs细胞染色体是否正常;成骨试剂盒检测hTERT-hBMSCs细胞向成骨分化的能力。 结果:骨髓间充质干细胞为贴壁生长的梭形细胞,表面抗原CD44表达阳性,CD34阴性。TRAP-ELISA法检测转染hTERT的hBMSCs细胞的端粒酶为阳性,而未转录的正常人的hBMSCs细胞则为阴性,说明转入hTERT基因后细胞端粒酶激活。hTERT-hBMSCs细胞比hBMSCs细胞增殖活跃,两组相比有显著的差异性,目前已传了11代,尚未见衰老迹象。染色体核型分析所示:hTERT-hBMSCs细胞染色体数目和结构正常,未见缺失、断裂、异位等畸变。成骨诱导结果表明hTERT-hBMSCs细胞仍具有向成骨分化的能力。克隆形成实验结果显示hTERT-hBMSCs细胞的克隆形成率较低,为0.66%,为正常细胞。 结论:导入外源性hTERT可激活细胞端粒酶的活性。hTERT基因转入细胞后,不影响其生物学特性和分化功能,仍然保持向成骨分化的能力。
[Abstract]:Objective: to construct immortalized human bone marrow mesenchymal stem cells. Methods: the viral plasmid pBabepuro-hTERT was introduced into the packaging cells by cationic liposome mediated method. The positive cell clones were screened by purine mycin (Puro) and the viral titer was determined. The supernatant hBMSCs cells of the culture virus were isolated and the transgenic cells were screened. We named them hTERT-hBMSCs cells. Telomerase activity of hBMSCs cells before and after transfection was detected by polymerase chain immunosorbent assay (ELISA) with telomerase repeat amplification (telomere repe at amplification protocol assay trap. The growth curve of hTERT-hBMSCs cells before and after transfection was measured by MTT method, and the effect of hTERT on cell proliferation was observed, and the proliferation ability of hTERT-hBMSCs cells was detected by clone formation assay. Chromosome karyotype analysis was used to observe whether the chromosomes of hTERT-hBMSCs cells were normal, and the ability of hTERT-hBMSCs cells to differentiate into osteoblasts was detected by osteoblast kit. Results: bone marrow mesenchymal stem cells (BMSCs) were spindle cells with adherent growth. The positive expression of surface antigen (CD44) was detected by CD34 negative. TRAP-ELISA was used to detect telomerase activity in hBMSCs cells transfected with hTERT, while hBMSCs cells in untranscribed normal persons were negative. The results showed that telomerase activation of hTERT-hBMSCs cells was more active than that of hBMSCs cells, and there was significant difference between the two groups. Chromosome karyotype analysis showed that the chromosome number and structure of human hTERT-hBMSCs were normal, and no aberrations such as deletion, breakage and ectopic were found. Osteogenic induction showed that hTERT-hBMSCs cells still had the ability to differentiate into osteoblasts. The clone formation rate of hTERT-hBMSCs cells was 0.66, which was normal. Conclusion: the transfection of exogenous hTERT can activate the telomerase activity of HTERT gene into cells without affecting its biological characteristics and differentiation function and maintain the ability of osteogenic differentiation.
【学位授予单位】:石河子大学
【学位级别】:硕士
【学位授予年份】:2007
【分类号】:R329
本文编号:2218872
[Abstract]:Objective: to construct immortalized human bone marrow mesenchymal stem cells. Methods: the viral plasmid pBabepuro-hTERT was introduced into the packaging cells by cationic liposome mediated method. The positive cell clones were screened by purine mycin (Puro) and the viral titer was determined. The supernatant hBMSCs cells of the culture virus were isolated and the transgenic cells were screened. We named them hTERT-hBMSCs cells. Telomerase activity of hBMSCs cells before and after transfection was detected by polymerase chain immunosorbent assay (ELISA) with telomerase repeat amplification (telomere repe at amplification protocol assay trap. The growth curve of hTERT-hBMSCs cells before and after transfection was measured by MTT method, and the effect of hTERT on cell proliferation was observed, and the proliferation ability of hTERT-hBMSCs cells was detected by clone formation assay. Chromosome karyotype analysis was used to observe whether the chromosomes of hTERT-hBMSCs cells were normal, and the ability of hTERT-hBMSCs cells to differentiate into osteoblasts was detected by osteoblast kit. Results: bone marrow mesenchymal stem cells (BMSCs) were spindle cells with adherent growth. The positive expression of surface antigen (CD44) was detected by CD34 negative. TRAP-ELISA was used to detect telomerase activity in hBMSCs cells transfected with hTERT, while hBMSCs cells in untranscribed normal persons were negative. The results showed that telomerase activation of hTERT-hBMSCs cells was more active than that of hBMSCs cells, and there was significant difference between the two groups. Chromosome karyotype analysis showed that the chromosome number and structure of human hTERT-hBMSCs were normal, and no aberrations such as deletion, breakage and ectopic were found. Osteogenic induction showed that hTERT-hBMSCs cells still had the ability to differentiate into osteoblasts. The clone formation rate of hTERT-hBMSCs cells was 0.66, which was normal. Conclusion: the transfection of exogenous hTERT can activate the telomerase activity of HTERT gene into cells without affecting its biological characteristics and differentiation function and maintain the ability of osteogenic differentiation.
【学位授予单位】:石河子大学
【学位级别】:硕士
【学位授予年份】:2007
【分类号】:R329
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